Epidemiology of Fascioliasis in Kermanshah Province, Western Iran.

Background
We aimed to determine the prevalence of fasciolosis in the definitive hosts (human and livestock) and intermediate (Lymnaea snails) hosts in Kermanshah Province, western Iran from 2014-2016.


Methods
The study on animals was descriptive and retrospective one. All daily records of animals slaughtered in the abattoirs were analyzed. For the study of human fascioliasis, 975 serum samples were collected from different parts of Kermanshah Province and analyzed using ELISA based on excretory-secretory antigens. Moreover, 4400 Lymnaea snails were collected from 25 habitats. The snails were identified and examined for presence of cercariae by shedding method.


Results
Fasciolosis was diagnosed in 1.7% of slaughtered animals, which was significantly greater than the other species (P<0.005). There was significant difference (P<0.001) between the prevalence of fasciolosis and seasonal pattern. As for human cases, five cases (0.5%) were positive for fascioliasis. Regarding the seropositivity to fasciolosis, no significant differences were found for age groups, sex, level of education and occupation. No Fasciola infection was seen in snails of the family Lymnaeidae.


Conclusion
The prevalence of Fasciola parasite was lower compared to other provinces. This is probably due to sequential decline in rainfall and hot climate that makes conditions difficult for the snail intermediate host snails and the larval stages of fasciolid trematodes. The habitual food of people is another important point.


Introduction
Fasciolosis is a parasitic disease caused by liver fluke species of the genus Fasciola, F. hepatica, and F. gigantica. This disease predominates in areas where livestock; normally sheep and cattle are raised, they being the most important reservoirs for the organism and causes economic loss due to condemnation of affected organs, fatality decreased production of meat, milk, and wool; decreased weight and infertility (1). Moreover, fasciolosis is an important zoonosis for public health with up to 2.4-17 million individuals thought to be infected worldwide and more than 180 million at risk of infection (2). Humans are accidental hosts by eating aquatic plants or drinking water contaminated with metacercaria (1). Infected ruminants can be a significant source of human Fasciola infection since the livestock grazing areas is often near to the freshwater plants grown for human consumption. The prevalence of fascioliasis in various livestock has been reported 0.1% to 91.4% in Iran (3 (4). However, in Iran, little information is available regarding the prevalence of Fasciola in intermediate hosts.
The first outbreak of human fascioliasis was reported in 1989 in Guilan Province (3). Since then various studies have been carried out on the prevalence of Fasciola infection in Iran (5)(6)(7)(8). In addition, minor emergence of fasciolosis was observed in Kermanshah, western province of Iran in 2000 (9). In Iran, human cases of infection with both Fasciola species have been previously reported by morphological and molecular surveys (7,10). In spite of this, it is difficult to distinguish Fasciola species under a microscope (11,12). At present, the prevalence of Fasciola infection in Kermanshah Province is still unknown. A lack of knowledge is the biggest hurdle on our understanding of the epidemiological characteristics and dynamic transmission of fasciolosis in this region. Therefore, the objective of the present study was to determine the prevalence of fasciolosis in the definitive host (human and livestock) and intermediate (Lymnaea snails) hosts in Kermanshah Province, west Iran.

Study area
Kermanshah Province, western Iran (lat 45.5° and 48° E, long 33.7° and 35.3° N), with an area of 24998 km 2 is composed of fourteen districts. The annual average temperature and rainfall are 26 °C and 1935 mm, respectively. The population of the province in 2017 was about 2 million (urban: 61.7%, rural: 37.7% and the rest were nonresidents) (Fig. 1). The province is located between Iranian Plateau and Mesopotamia Plain in the mountainous area and Zagross heights along with summits to cover the whole area. Parts of the slopes of a lower incline and mountainous expansions are low lands and alluvium plains. The climate of the highlands is mild in summer and cold in winter, with heavy snowfall; only the province's western strip belongs to the warm climate.

Ethical approval
This study was approved by the local Ethics Committee of Tehran University of Medical Sci-ences. The informed consent of the participants or their guardians was obtained.

Serological examination
Overall, 975 blood samples were collected from selected subjects by randomized cluster sampling method. Following centrifugation, sera were stored at -26 ºC until tested. A questionnaire was used to obtain demographic information such as age, sex, education, occupation and consumption of local freshwater vegetables. The serum samples were analyzed using ELISA method performed as previously described (7). Plates were read on by ELISA reader at an absorbance of 490 nm.

Identification of Fasciola larval forms in Lymnaea snails
During the period from Sep 2014 to Oct 2016, 4400 Lymnaea snails were collected from 25 habitats (agriculture, rivers, swamps, and various streams) in the study area. Snails were first identified based on the morphological keys (4). Then each 7 to 8 snails were placed in a petri dish containing water and were placed against light for two hours or overnight in the room temperature. The snails examined for presence of cercariae by shedding method (the release of cercariae from the host snail in nature). Petri dishes were checked carefully under a dissecting microscope for the presence of Fasciola larvae. The cercariae were identified based on a key for identifying trematode cercariae.

Slaughterhouse Data
We used all daily condemnation records from 178920 livestock (cattle, goats, and sheep) slaughtered in the abattoirs of different localities in Kermanshah, from Sep 2014 to Oct 2016. Fascioliasis was recognized by morphological characteristics of them. Data, such as species, gender and season, were recorded on standardized data sheets.

Statistical analysis
Statistical analysis was done using SPSS (ver. 16, Chicago, IL, USA). Chi-square test was used for analyzing data. Cut-off was calculated as mean± 3 SD.

Results
During the period under study, 178920 animals (cattle 41805, sheep 125313 and goats 11802) were slaughtered in the abattoirs in Kermanshah Province. Overall, the prevalence of ruminant fasciolosis was 1.7%. Fasciolosis was significantly higher (P<0.05) in cattle (3%) than sheep (1.1%) and in goats (2.3%) ( Table 1). There was significant difference (P<0.01) between the prevalence of fasciolosis and seasonal pattern. The highest prevalence rate was in the autumn (Table  2).  and 91 (2.1%) L. palustris. Fasciola infected snails were not found at Kermanshah Province (Fig. 2).  Figure 3 shows the distribution of optical density (OD) in subjects and healthy control cases. Cutoff was calculated as 0.2. Five cases (0.5%) were positive for fascioliasis by ELISA test (Fig. 3). The seroprevalence of fascioliasis among females was 0.5% and 0.36% in males. The highest posi-tive rate was in age group of 20-29 yr old. According to job, three were housekeepers, one student, and one self-employed. The prevalence of human fasciolosis in rural and urban areas was 1.73% and 0.24% respectively. Three of seropositive cases used to eat raw vegetables.

Discussion
Fascioliasis is a significant limiting factor on the economy of the livestock industry and human health; therefore, the development of control strategy to reduce the incidence is a priority. Now, 17 years after the first epidemic human fascioliasis was reported (9), little information is available regarding the prevalence of Fasciola infection in Kermanshah Province. The results of the presented study provide an overview of the occurrence of Fasciola spp. in this province.
In this study, the prevalence of Fasciola parasite in slaughtered animals was 1.7%, which were relatively lower compared with previous researches in which those in Jahrom and Lorestan were between 3.44% and 6.3%, respectively (13,14). In old reports, the prevalence of livestock fascioliasis has been reported 0.1% to 91.4% in Iran (3,15). Although molecular identification of Fasciola species was not carried out in this study, F. hepatica seems to be the major cause of fascioliasis in animals in this region (16).
The overall human seroprevalence as determined by ELISA was 0.5% that was lower than some studies previously conducted in some country provinces of Lorestan (1.3%) (7), Meshkin-Shahr (1.96%) (6), Isfahan (1.7%) (8), Tehran (24.8%) (5); and higher than in others such as Chaharmahal and Bakhtiyari (0.1%) (17). However, serological results may detect active or past infection because high antibody titers may remain in the human body for long period after treatment and cure. One of the possible reasons for the decrease the prevalence infection is sequential decline in rainfall, hot climate and dried the ponds in the area in recent years that could have a significant impact on the survival of both the snail intermediate host snails and the larval stages of fasciolid trematodes (18

Conclusion
Although serology blood test showed very low infection rate (0.5%) but the presence of intermediate hosts and animal reservoirs might be suggested much greater infection in these areas. However, further research on the correct identification of intermediate-host species, as well as possible routes of transmission of trematode infection to humans, is needed.

Ethical considerations
Ethical issues (Including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc.) have been completely observed by the authors.